This invention relates to optical scanners suitable for analyzing images on a film. For example, such scanners are suitable for analyzing an autoradiographic image of the products of a deoxyribonucleic acid (DNA) sequencing reaction in order to determine the nucleotide base sequence of a DNA molecule.
DNA is a double stranded or single stranded polymeric molecule formed by covalent joining of the four deoxynucleotide bases, namely, adenine (A), guanine (G), thymine (T), and cytosine (C). The sequence of these deoxynucleotide bases determines the utility of a DNA molecule. For example, a DNA molecule may encode a controlling region for another DNA molecule, or may encode a polypeptide or a protein. It is common to determine the deoxynucleotide base sequence of a DNA molecule in a variety of manners. Generally, this process, called DNA sequencing, involves generation of four populations of single-stranded DNA fragments having one defined terminus and one variable terminus. The variable terminus generally terminates at a specific given nucleotide base. The four different sets of fragments are each separated, on the basis of their length, to form bands on a high resolution polyacrylamide gel. Each band on the gel corresponds colinearly to a specific deoxynucleotide base in the DNA sequence, thus identifying the position in the sequence of a given deoxynucleotide base.
The most commonly use method of DNA sequencing is termed dideoxy-sequencing. This method involves enzymatic synthesis of a DNA strand using a DNA polymerase. Generally, four separate synthesis reactions are run, each reaction being caused to terminate at a specific nucleotide base by incorporation of an appropriate chain terminating agent, e.g., a dideoxynucleotide. Enzymes used for dideoxysequencing include the E. coli DNA polymerase large fragment (Klenow), reverse transcriptase, T7 DNA polymerase, and Tag DNA polymerase.
Tabor and Richardson 86 Proc. Natl. Acad. Sci. USA 4076, 1989 describe use of a manganese-based buffer (in place of the usual magnesium-based buffer) for a DNA sequencing reaction. Use of manganese results in uniform terminations of DNA sequencing reactions with intensity of adjacent bands on a polyacrylamide gel varying in most instances by less than 10%. They state that this property should be of use for DNA sequencing, particularly by automated procedures.
The normal nomenclature used by persons in this field will be used in this application. Thus, the term "lane" refers to a generally narrow linear band between the top and bottom of an acrylamide gel in which a sample to be electrophoresed is placed and then electrophoresed. A "base" is generally used to refer to a deoxynucleotide base. A "smile" refers to an artifact produced during electrophoresis where the distance traveled by samples within adjacent lanes differs, such that the central lanes in an acrylamide gel will travel further than those on either side, thus creating a curved front in the gel. A "band" refers to the image on an autoradiogram of a DNA product running within a lane at a specific linear position along that lane. DNA products which differ by one or more bases form distinct bands along a lane. In ideal conditions, a band is a rectangular square the width of which is equal to the width of a lane, and the length of which is relatively small such that adjacent bands do not merge.
There follow references to machines designed to aid analysis of products of a DNA sequencing reaction, for example, an autoradiogram resulting from a DNA sequencing reaction. These references are generally based on trade literature. These machines are not admitted to be prior art to the present application.
IntelliGenetics.TM. literature describes a device for acquiring the image of a DNA sequencing autoradiogram and deciphering the pattern of bands. It is described as being capable of compensating for common defects. For example, it is said to straighten distorted lanes, flatten bands, and minimize smiles even in the same lane set.
UltroScan.TM. literature is a laser densitometer suitable for scanning autoradiographs, photographs, slab gels, blots, and tube gels. Slab gels and tube gels refer to agarose or polyacrylamide gels formed, respectively, as a flat rectangular gel or within a tube. The literature states that "only a laser can help . . . get the maximum amount of information from today's high resolution separations . . ."
BioRad.TM. literature describes a video densitometer for analyzing autoradiograms and gels. The system is provided with a 1728 element CCD linear diode array detector, and a light source for transmission or reflectance which illuminates a narrow 20 cm slot. Light from the sample is directed by two mirrors to a lens which focuses the light onto a CCD linear array detector. 1660 elements of the linear array are used to measure the data simultaneously. The sample is placed on a platten controlled by stepper motor. The platten is moved back and forth either automatically or manually. An entire lane of data is illuminated and measured at once.
BioMed Instruments.TM. literature describes several laser scanning densitometers. A ribbon laser beam suitable for scanning by absorbance or transmittance, is used scan two dimensional gels.
Applied Biosystems.TM. literature describes a sequencing system using four different fluorescent dye labels each corresponding to one of the four deoxynucleotide bases. The gel is analyzed by a laser light beam which scans the electrophoresis gel.
Molecular Dynamics.TM. literature describes a densitometer able to automatically scan an entire sample area. 13,000 density measurements are made on each square centimeter of sample.
Hitachi.TM. America literature describes a DNA film scanner. The scanner appears similar to a desk top copier and uses a charge coupled device (CCD).
BioRad.TM. literature describes an automatic sequence reader and analysis system. The optical scanner utilizes a charge coupled device. The software is designed to correct for smiling and other electrophoretic artifacts.
BioImage.TM. literature describes an instrument able to scan whole gels quickly and automatically. The gel is scanned using a Kodak camera or an optional laser scanner. The camera is a Kodak CCD camera, formed as a two dimensional CCD device having 1024.times.1024 arrays. Reflective scanning is also possible.